The present invention relates to synthetic membranes, micelles, and vesicles.
In living plant and animal cells, non-polar lipids are stored in droplets within the cytoplasm and are rarely found in biological membranes. Proteins which have been isolated from membranes of living cells often possess a sequence of non-polar amino acids which anchor the proteins through hydrophobic associations within the interior of phospholipid bilayers. Other proteins are anchored through covalent bonds to glycophospholipids. The oligosaccharide moieties of membrane glycoproteins and glycophospholipids project into the aqueous environment.
Phospholipid monolayers and bilayers form micelles and liposomes which have been used successfully in delivering pharmaceutical agents. However, the chemical and mechanical instablity of these constructs have posed problems. Liposomes are prone to oxidation and tend to aggregate and fuse during prolonged storage. Injected liposomes are degraded by the lecithin-cholesterol acyl transferase of high density lipoproteins, and are cleared from the bloodstream by macrophages and hepatocytes. Though it is possible to attach certain glycoproteins to phospholipid micelles, polar phosphate heads facing the aqueous solution tend to inhibit contact of lipid tails with the hydrophobic amino acids of glycoproteins.
Both amino acid sequences and oligosaccharide segments of glycoproteins can contribute chemical and biological properties that may be useful components of drug delivery agents. Glycans present on the exterior surfaces of lipo-glycoprotein vesicles and micelles may be useful for protecting ingested therapeutic and nutritional substances against chemical degradation in the gastrointestinal tract. For instance, Gu et al. demonstrated the role of oligosaccharide moieties in protecting ovomucoid, a glycoprotein found in hen egg whites, against tryptic hydrolysis and heat denaturation.
Sialic acid, the terminal sugar of many oilgosaccharides produced in animal tissues, is ionized at pH 7 (Lehninger et al., 1993). Its presence inhibits uptake and degradation by hepatocytes of circulating blood cells and glycoproteins. Baenziger et al. discovered in 1992 that terminal sulfated oligosaccharides also prevent hepatocyte clearance from the circulation. The presence on micellar surfaces of glycoproteins bearing terminal sialic acid or sulfated glycans may thus prevent or delay uptake by the liver and phagocytic blood cells of therapeutic agents injected into the bloodstream.
Micelles are aggregates of substances in which hydrophilic polar groups of compounds orient themselves toward and interact with the aqueous phase. The hydrophobic nonpolar hydrocarbon chains of the micelles are hidden within the structure. For example, micelles which contain soap molecules remain evenly suspended in water because their surfaces are negatively charged and the micelles repel each other. Micelles prepared from phospholipids and oligosaccharide-lipid complexes have been used to prepare vaccines using natural and synthetic oligosaccharides, which are immunogens, to prepare stabilized vaccines, disclosed in U.S. Pat. No. 5,034,519, the entire contents of which are hereby incorporated by reference.
It is also known that amphipathic proteins such as cytochromic oxidase, an intrinsic enzyme found in mitochondrial membranes, when placed in suspension with lipids form sac-like vesicles that are, in effect, man-made membranes. These vesicles have been used as model systems for the study of the isolated protein""s relationship with lipid bilayers.
Compans, U.S. Pat. No. 4,790,987, teaches the preparation of viral glycoprotein subunit vaccine by complexing a lipid with the glycoprotein. Compans also teaches that the complexes can be obtained by dissolving a lipid in a dialyzable detergent solution containing glycoproteins, then dialyzing the solution to obtain the protein-lipid complex. The lipids are phospholipids. The resulting complexes are then administered in pharmaceutically acceptable carriers.
Rutter et al., in U.S. Pat. No. 4,769,238, note that vaccine bound to a membrane may be superior to non-membrane bound proteins.
Mouritsen et al. have studied protein-protein and protein-lipid interactions in phospholipid bilayers in an attempt to refine the fluid mosaic model of biomembranes proposed in 1972 by Singer-Nicolson. In 1995 Oin et al. produced two-dimensional crystals of avidin on the hydrophobic surface of a phospholipid monolayer. In both synthetic and biological phospholipid membranes, polar heads of component molecules lie adjacent to one another at the aqueous interface, shielding their hydrophobic lipid tails from contact with water or molecules dissolved therein.
Unger et al., in U.S. Pat. No. 5,853,752, described methods of preparing gas-filled phospholipid microspheres to act as contrast agents in ultrasound image formation. Feinstein, in U.S. Pat. Nos. 4,718,433 and 4,774,958, teaches the use of albumin-coated microbubbles for the same purpose. These technologies exploit the changes in acoustic impedance that occur as sound waves encounter interfaces between solids, liquids, and gases. The greater the elasticity of this interface, the more efficient the reflection of sound. Changes in acoustic impedance result in a more intense signal in the ultrasound image.
In U.S. Pat. No. 5,846,744, Athey et al. describe a novel sensor format based on the impedance analysis of polymer coatings on electrodes for determining the presence or amounts of an analyte in a sample of assay medium.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to provide micelles and vesicles which can be used for transporting substances that can be dissolved or suspended in lipids.
It is a further object of the present invention to extract glycoproteins from solution.
The present invention provides synthetic membranes, micelles and vesicles that form in response to the spontaneous orientation of hydrophobic and hydrophilic groups in aqueous media. Unlike phospholipid monolayers, micelles, and liposomes, the membranes of the present invention comprise a sheet of glycoprotein molecules associated on one face with a non-polar liquid, and on the other face with water. The oligosaccharide moieties of glycoprotein molecules are oriented toward the aqueous environment.
The micelles of the present invention are relatively stable in aqueous environments, and thus are useful for transporting substances that can be dissolved or suspended in lipids for inclusion in the micelles. Lipo-glycoprotein micelles are also useful for protecting substances contained within the hydrophobic compartment from dispersal or degradation until the micelle structure is disrupted.
Certain glycoproteins incorporated into the membranes of micelles according to the present invention may limit the first pass effects of drugs contained within the hydrophobic compartment by resisting chemical degradation in the stomach or other segment of the gastrointestinal tract through which they pass. Other glycoprotein components of micelles employed in the delivery of injected drugs may serve as signals for inducing or inhibiting their uptake by cells of certain tissues. Still other glycoproteins contain amino acid sequences or segments of oligosaccharide moieties that are bioactive and may be used as therapeutic agents. Micelles of the present invention can also be used for delivering such glycoproteins. For instance, oligosaccharide components of a membrane may confer to drug delivery agents properties such as cellular recognition signals. It is also possible to include two or more species of glycoprotein with differing biological activity on a single micelle. In addition, membrane surface characteristics and micellar diameter can be used to prevent the movement in the body of bound glycoprotein from one fluid compartment to another.
Lipo-glycoprotein membrane formation can be used to extract both glycoproteins and oils from aqueous solution. During the process of membrane formation, strong intermolecular attraction traps glycoproteins and hydrophobic liquids. Very small concentrations of glycoprotein and oil can be collected in membranes and removed mechanically from the surface of an aqueous solution. Volatile organic liquids are sequestered within micelles that form when the membrane is agitated.
Membrane formation can also be employed in sensor design. For example, an oil-filled tube having a small aperture can be inserted into an aqueous solution, then withdrawn and subjected to measurement of light absorption or penetration through the aperture to detect proteins present therein. To sense the escape of organic solvents from an industrial process, a cool sample can be introduced into an aqueous solution of glycoprotein and agitated. The formation of micelles at the surface would not only indicate the presence of hydrophobic chemicals, but would facilitate their recovery.
Membranes or micelles bearing glycoprotein antigens or antibodies can also be used to extract target molecules from aqueous samples of solutions containing tissue fluids or disease-causing organisms. Sensors employing this method might be useful in the inspection of foods and in forensic science. Filters made of cellulose or synthetic fibers coated with lipo-glycoprotein membranes bearing antibodies might also be used to remove certain organisms or biotoxins from aqueous solution. Enzymes can be incorporated into the micelles of the present invention to catalyze a variety of chemical reactions in aqueous media.
Hydrated glycans are preserved on membrane surfaces and can be studied in situ. The association of lipid with glycoproteins appears to maintain the orientation of carbohydrate moieties and facilitate the rehydration of dried micelles. Oligosaccharide components prevent aggregation and fusion of micelles and inhibit their degradation by heat and protease.
Stable glycoprotein membranes are prepared by first layering a non-polar liquid on top of an aqueous mixture containing at least one glycoprotein or proteoglycan. A membrane then forms at the aqueous-non-polar interface site. When agitated, said membrane breaks apart into vesicles and micelles having an oligosaccharide surface facing the aqueous solution and enclosing one or more hydrophobic substances. Gentle agitation of the membrane produces vesicles of sufficiently large diameter to permit easy removal from the aqueous solution in which they are disposed. The large vesicles may then be passed through a strainer or screen to divide them into smaller micelles. Micelles produced by association of non-polar liquids with glycoproteins are mechanically and chemically more stable than those derived from phospholipids.
An inverse micelle may be produced by introducing a droplet of aqueous solution containing glycoprotein into a non-polar liquid such as olive oil. The glycoprotein forms a membrane around the aqueous solution, creating a vesicle having a hydrophobic exterior surface. Inverse micelles introduced into the lipid layer floating above a lipo-glycoprotein membrane will be enclosed within the hydrophobic compartment when the membrane is agitated to form closed vesicles.